There are numerous disease states in humans which are characterized by a high level of bone resorption and/or an abnormal balance between bone formation and bone resorption. Among the more common of these are osteoporosis, osteoarthritis, rheumatoid arthritis, and conditions related to the progress of benign and malignant tumors of the bone and metastatic cancers that have migrated to bone cells from elsewhere in the body, e.g., from prostate or breast initial tumors. Other conditions associated with changes in collagen metabolism include osteomalacial diseases, rickets, abnormal growth in children, renal osteodystrophy, and drug-induced osteopenia. Further, abnormalities in bone metabolism are often side effects of thyroid treatment and thyroid conditions per se, such as primary hyperparathyroidism and thyrotoxicosis as well as Cushing's disease.
The organic matrix of bone consists of approximately 90% of type I collagen, which contains two .alpha.1 and one .alpha.2 chain coiled around each other to form a triple helix. The helical domain of each chain is preceded by a short N-telopeptide and is followed by a short C-telopeptide. The biosynthetic pathways leading to formation and maturation of Type I collagen have been described (e.g, Alberts et al., 1989; Eyre, 1987). Collagen chains are initially synthesized as procollagen chains which associate to form trimeric, triple helical complexes. Following secretion into the extracellular space, the trimeric molecules are cleaved to release the propeptides from the N- and C-termini to form collagen (also known as tropocollagen) molecules. The collagen molecules associate to form rod-shaped fibrils in which the molecules are packed in parallel, staggered arrays. Formation of various intra- and intermolecular crosslinks within and between adjacent collagen molecules impart increased stability.
In mammals, the formation and maintenance of bone collagen tissues is understood to be a dynamic process mediated by bone forming cells (osteoblasts) and bone degrading cells (osteoclasts). An imbalance between the rates of bone formation and bone degradation, and particularly elevation in bone degradation, can result in serious pathological conditions deleterious to health.
Over the past several decades, various methods for diagnosing or monitoring abnormalities of bone collagen degradation have been proposed. For example, hydroxyproline, a major constituent of collagen polypeptides, was proposed as a possible marker in urine many years ago. There are, however, several disadvantages with the use of measurement of hydroxyproline as a marker, including the need for a lengthy acid-hydrolysis step, a lack of specificity for Type I collagen (from bone), and the substantial metabolism of free hydroxyproline in the liver. In summary, hydroxyproline has been rejected as a clinical marker for bone resorption conditions.
Hydroxylysine and certain glycosylated forms thereof have been proposed, but use of these markers has been limited due to the lack of a convenient assay method suitable for general clinical use, as well as by the need for an acid hydrolysis pretreatment step, like hydroxyproline.
Certain terminal fragments of collagen have also been investigated for possible diagnostic applications. Prior to the applicants' present invention, it was generally believed in the art that peptides originating from the helical regions of collagen could not be used as indicators of bone resorption because of substantial degradation caused by endogenous proteases in the extracellular space, blood circulation, and urine. Accordingly, practitioners in the art have focused on measuring fragments from the telopeptide regions of collagen that contain crosslinking species such as pyridinoline crosslinks, which presumably protect associate collagen chain regions from proteolytic degradation (e.g., Risteli, 1992). Methods for measuring such telopeptide fragments have been described, for example, in WO 89/04491 (Eyre), WO 95/08115 (Qvist), and WO 94/14844 (Baylink). Methods have also been described for measuring telopeptide crosslinks themselves, without attached collagen chains (see Robins, WO 91/10141; Cerelli et al., WO 94/03814; and Kung et al., WO 94/14072).
Telopeptide fragments have been reported to be useful in connection with bone resorption conditions. However, procollagen peptide species have been reported to be useful for measuring of bone formation (e.g., Taylor, 1994), a process that is irrelevant to most bone resorption-related diseases.
Accordingly, there remains a need to develop new markers which are useful for diagnosing and monitoring abnormal bone resorption conditions. Ideally, such a marker should be measurable in biological fluids such as urine and serum, conveniently by immunoassay. The marker should be useful in diagnosing the presence of bone resorption disorders which are characterized by above-normal levels of bone degradation. In addition, the marker should be useful for detecting changes in the status of bone degradation in the subject over time, particularly in response to therapeutic treatment.